UNITED ST ATES ENVIRONMENT AL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

AUG 2 4 2015 

OFFICE OF CHEMICAL SAFETY 

AND POLLUTION PREVENTION 

MEMORANDUM 

UBJECT: 

FROM: 

PEER 

REVIEW: 

TO: 

Environmental Risk Assessment for a FlFRA ection 3 Limited Seed Increase 

Registration of DvSnf7 Double Stranded RNA (dsRNA) and Cry3Bb I Bacillus 

thuringiensis Derived Insecticidal Protein as Ex pressed in MON 87411 Maize: 

EPA File Symbol 524-ARI; PC Codes: 006566, 006580; Decision o. 487782; 

Submission os. 960 120, 963404, and 94746 1; DP Barcodes: 4279 17, 424378, 

426505, and 420443 ; MRI Ds 4931 5 1-11 through -19. 493 15 1-2 1 and -22, 

495058-02, -03, and -05, 495533-04 and -06: ubmitted by Monsanto Company 

Shann

Microbi

on 

al 

B

P

orges, 

esticide

Lead 

s Bran

Bio

c

lo

h 

gist ~~~ 

Biopesticides and Pollution Prevention Division _ 'Jr' ... t 

Chris Wozniak. Ph.D., Biotechnology Special Assistant L j - C /{~ ~ Biopcstic ides a nd Pollution Prevention Di vision l'v'-" 1 L/ 

Jeannine Kausch, Regul atory Action Leader 

Microbial Pesticides Branch 

Biopcsti cides and Pollution Prevention Division 

I. Introduction 

Monsanto Company has submitted an application for a FJFRA Section 3 seed increase 

registration for l\'>'0 new plant incorporated protectant (PIP) active ingredi ents expressed in Event 

MO 874 I 1 transgeni c corn. The new acti ve ingredients include a double stranded RNA 

(dsR A) transcript (DvSnf7) derived from western corn rootworm (Diahrotica virg(fera 

virf?((era, WCR) and also a Cry3 Bb 1 insecticidal protein deri ved from Bacillus thuringiensis (Bt) 

subsp. kumamotoensis. The DvSnJ7 dsRNA and Cry3Bbl protein expressed in MON 874 11 

confer resistance to corn rootworm (Diabrotica spp., CRW). MON 87411 also expresses the 5-

enolpyruvylshikimatc-3-phosphate synthase (EPSPS) protein, which confers tolerance to 

pho~ 

:;J 

The DvSnJ7 dsRNA results from expression of an in verted repeat sequence designed to match 

the sequence of WCR Sn.f7 gene. Expression of the sequence results in the formati on of the 

dsRNA transcript containing a 240 bp fragment of the WCR Sn.f7 (DvSnt7). Snt7 is a vacuolar 

1 
sorting protein belonging to the Endosomal Sorting Complex Required for Transport (ESCRT)-

III complex. which is involved in sorting of transmembrane proteins en route to lysosoma l 

degradation through the endosomal-autophagic pathway in a number of organisms. Once 

consumed, the DvSnt7 dsRNA is recognized by the CR w·s RNA interference (RNAi) 

machinery wherein it is cleaved into 2 1-24 mer small interfering RN As (siRNAs). These 

siR As bind to a RNA induced silencing complex (RISC), which ultimately leads to downregulation of the targeted DvSn.f7 gene and mortality. 

The Cry3Bb 1 protein expressed by MO 87411 is a delta-endotoxin from Bacillus thuringiensis 

subsp. kumamotoensis, whi ch has specific activity against insects within the Order Coleoptera. 

EPA determi ned that the Cry3 Bb I insecticidal protein expressed by MON 874 11 is 

biochemicall y and functionally equivalent to the Cry3Bb l expressed in MON 88017 com (U.S. 

EPA 2015). The activity of the Cry3Bbl protein expressed in MON 88017 was also determined 

to be biochemically and functionally equivalent to Cry3Bbl expressed in MON 863 maize, and 

both were determined to have no unreasonable adverse effects on nontarget organisms (U. 

EPA 2010a). 

An Experimental Use Permit (EUP) was previously issued for MON 87411 (and a similar PIP. 

MON 874 10) and other previo usly registered PIPs in 20 13, ending in 20 15. This EUP involved 

approximately 13,000 acres on which MO 8741 1 and MON 87410 were to be grown, and the 

duration of the EUP was two years. This EUP was extended in 20 14 until February 2016. and the 

acreage involving MON 87411 was expanded to approximately 19,000 acres (MON 87410 was 

no longer being tested in the EUP). The EUP was extended again in February 20 15, but the 

acreage involving MO 87411 was reduced to approximately 9,500 acres. The ecological risk 

assessment for this EUP relied primarily on data demonstrating the specificity of the DvSnf7 

dsRNA for the WCR target pest as well as rationale describing some barriers to uptake of the 

DvSnf7 dsRNA in nontarget organisms. In light of the limited acreage involving MON 87411 

and MON 87410 and the limited duration, EPA determined that the risk of adverse e ffects to 

populations of nontarget organisms was minimal. 

Due to uncertainties of dsRNA pesticide acti ve ingredients identified in the literature and 

potentially associated with human and nontarget organism risks. EPA consulted with the FIFRA 

Scientific Advisory Panel (SAP) for guidance in understanding and addressing these 

uncertainties. The meeting of the SAP was held January 28, 2014, with minutes published in 

May of that year 1

• The SAP consultation was held independentl y of any registratio n involving a 

dsR A based pesticide, including MON 87411. Monsanto Company has included their response 

to the minutes of the SAP in the information submitted to support this registration (MRID 

49505801 ). 

The initial application for MON 87411 submitted in 2014 was for a seed increase registration 

with the standard limits of 20.000 acres per county and up to a combined U.S. total of 250,000 

acres per Pl P active ingredient per registrant per year. In light or uncertai nties discussed at the 

SAP meeting, EPA issued a 75-day de ficiency letter in December 2014 requesting additional 

data. In February 2015. Monsanto requested a change to the proposed registration by limiting the 

registration to two years with 15,000 acres of MON 874 11 grown in each year. A response to the 

1 l111p. \\ \\'\\ .cpn.;.?.ov ~t:ipol\' sap meeting,~ 20 I..\ i<muan 'O 128 14111i11111es.pdr 

2 
75-day letter (MRlD 49553302) was also submitted, which contained some additional data, as 

well as rationale supporting waivers for the additional data requested. 

This memorandum contains the environmental risk assessment for OvSnl7 and Cry3 Bb I 

expressed in MON 87411 for the proposed seed increase registration. Data submitted for Dv nf7 

and data bridged from a previous registration of Cry3Bb1 (see discussion below) are considered 

in the context of environmental exposure relevant to expression of these active ingredients in 

MON 87411. This information and additional lines of evidence are used to characterize risk for 

nontarget organisms under the limited registration described above. Pertinent information from 

the response to the 75-day Jetter and the response to the minutes of the AP are included in this 

risk assessment. 

II. General Approach to Ecological Risk Assessment for PIPs 

EPA·s current ecological risk assessment approach for PlPs was developed primarily from 

experience with Br-derived Cry and Yip proteins. These proteins are generally understood to be 

specific to target pests and related insects within the same taxonomic order, and with nearl y two 

decades of history indicating safe use. EPA considers the current approach sufficient for 

determining ecological risks of Br-derived protein PIPs, including the Cry3 Bb I protein expressed 

in Event MON 87411 corn. 

This approach is described in several Biopesticidc Registration Action Documents (BRADs) for 

Cry or Yip proteins (e.g., see U.S. EPA 20 10a, pages 65-69). To summari ze, the approach 

consists of a ti ered testing scheme (Tiers I - IV) focusing on detennination of hazard, and testing 

is based o n the data requirements for microbial pesticides published under 40 CFR 158.2 150 and 

885 and 850 series OCSPP Harmonized Guidelines. At Tier I, studies are designed to be 

simplified and to estimate of hazard of several non target laxa under "worst-case" exposure 

conditions. A lack of adverse effects under these conditions would provide enough confidence 

that there is no risk and no further data would be needed. Screening (Tier 1) maximum hazard 

dose tests are conducted at exposure concentrations several times higher (e.g., > 1 OX when 

possible) than the highest concentrations expected to occur under realisti c field exposure 

scenarios, \vi th mortality as the toxicological endpoint. A threshold of 50% mortality is used as a 

trigger for additional higher-tier testing. When screening studies suggest potentially unacceptable 

risk additional studies are designed to assess hazard under mo re realistic fi eld exposure 

conditions. Tiers II - IV generally encompass definitive hazard level determinations, chronic 

studies, and longer term greenhouse or fi eld testing, and are implemented when unacceptable 

e ffects arc seen at the Tier I screening level. 

When screening tests indicate a need for additional data, the OCSPP Harmonized Guidelines call 

for testing at incrementally lower doses in order to establish a definiti ve LDso and to quantify the 

hazard. A risk dete rmination is made by comparing the toxicological endpoint to the estimated 

environmental concentration (EEC). 

In additio n to the toxic ity data, additional data are also considered regarding the environmental 

persistence of Pl Ps. as well as the potential for gene flow and development of weedi ness. EPA 

requires laboratory data demonstrating the degradation of the PIP in soils typical of agronomic 

3 
areas where the PIP crop is grown. To assess gene flow and potential for development of 

weediness, EPA considers several lines of evidence related lo characteristics of the crop plant, 

including reproduction, presence of wild relatives, and containment or other mitigating measures. 

Because of uncertainties associated with the potential for unexpected effects related to exposure 

to dsRNA, EPA raised questions to the SAP regarding the applicability of the above approach lo 

dsRNA PIPs. The SAP found that this approach was not sufficient to determine risks to nontargct 

organisms, and suggested an alternative framework (see pages 61-64 of the SAP minutes), whi ch 

EPA is currently evaluating. 

Ill. Nontarget Organism Exposure Assessment 

Two separate SAP reports (October 2000 and August 2002) recommended that non-target testing 

of PIPs (in those cases. Bt deri ved proteins) should focus on invertebrate species exposed to the 

crop in which the PIP(s) will be expressed. EPA has previously determined, based on these 

recommendations, that non-target organisms with the greatest exposure potential to PIPs in 

transgenic corn fields arc beneficial insects that feed on plant tissues (e.g., pollen), so terrestrial 

invertebrates are the focus of non target organism testing and of PIP risk assessments. 

Nonetheless, EPA has recognized that exposure to other non target organisms can occur (e.g., 

exposure to birds by consumption of pest insects that have consumed the PIP), and has required 

testing on representative species. 

The EPA risk assessment is centered on determination of hazard at the field exposure rates, 

which are typically based on expression levels within the plant for PIPs. For the purposes of the 

non-target organism risk assessment ofDvSnf7 and Cry3Bbl in Event MON 8741 I com, 

estimated exposures are based on the highest concentration of these active ingredients expressed 

in com tissue(s) based on protein expression studies (MRI Os 493 I 5 104 and 49315105). Fresh 

weight and dry weight measurements of protein expression per gram of tissue are typically 

determined for PIPs, although estimates of exposure are based on dry weight measurements, as 

fresh we ight measurements can be highly variable, as confirmed by the SAP in October 2000. 

Mean levels of Cry3Bb I expressed in various tissues of MON 87411 (MRID 49315105) ranged 

from 4 μg Cry3 Bb I lg tissue dry weight (dwt). as expressed in grain, to 340 μg Cry3 Bb Ilg tissue 

dwt. as expressed in the whole plant sampled during the Y3-V4 development stage. Ranges or 

mean expression levels were 170 - 270 ~L g/g dwt in leaves, 75 - I 80 μgig dwt in root, 120 - 340 

μgig dwt in whole plant, and were measured at 2 1 μgig dwt in stover, 4 μgig dwt in grain, and 36 

μg/g in pollen. Senescent root, forage, and forage root had concentrations of I 9, 39, and 36 μg/g 

dwt of Cry3 Bb I, respectively. Leaf, root, and whole plant were sampled at the Y3-Y4, V6-V8. 

V I 0-Y I 3, and V 14-R I development stages, and expression generally decreased over the 

sampling periods. The highest expression reported was 460 μgig dwt, which was the maximum 

of the range of expression levels measured for whole plant. The expression levels of Cry3 Bb I in 

MON 874 I I corn plant tissues are comparable those of the Cry3 Bb I expressed in MON 863 

corn, and are lower than mean dry weight expression levels ofCry3Bbl in MON 88017 corn 

(U .S. EPA 2010a). 

4 
Ex pr1.;ssion of DvSnt7 dsRNA was similarly determined in MON 874 11 (MRlD 493 15104). 

Ex pression leve ls were very low, and mean expression levels ranged from 0.1 34 x I 0-3 μ g 

Ov nt7 dsRNNg tissue dwt, as determined in pollen, to 84.8 x I o3 μg DvSnf7 dsRNN g tissue 

dwt in whole plant sampled during the V3-V4 development stage. Ranges of mean expression 

levels were 44.6 x 10-

3 - 73.9 x I 0-3 μ g/g dwt in leaves, 6.84 x 10-3 - 23 .9 x 10-

3 μ gig dwt in root, 

18.5 x I 0-3 - 84.8 x I 0-3 μgig dwt in whole plant, and were measured at 0.677 x I 0-3 μgig dwt in 

stover, 0.104 x 10-

3 μgig dwt in grain, and 0.1 34 x I 0-3 μ g/g dwt in pollen. Senescent root, 

forage, and forage root had concentrations of 1.39 x ·10-

3, 4.26 x I o3, and 2.37 x 10-3 μg/g dwt, 

respectively. As with Cry3Bbl, leaf, root, and whole plant were sampled at the V3-V4, V6-V8, 

VI 0-V 13, and V 14-Rl development stages, and expression generall y decreased over the 

sampling periods. The highest expression reported was 2 13 x I 0-3 μgig dwt, which was the 

maximum of the range of expression measured for whole plant. 

IV. Nontarget Effects Data 

A. Overview of Data Submitted 

In the absence of PIP-specific risk assessment guidance, EPA has historically required applicants 

for Pl P registrations to meet the 40 CFR Part 158 data requirements for microbia l pesticides. 

These requirements include testing on birds, mammals, nontarget insects, honey bee, plants, and 

aquatic animal species. The October 2000 SA P recommended that while actual plant material is 

the pre ferred test material, bacteria-derived active ingredi ent is also a valid test substance, 

parti cularl y in scenarios where test animals do not normally consume corn plant tissue and where 

large amounts of active ingredi ent are needed for maximum hazard dose testing. In support of 

the MO 87411 registrations, Lest substances used in the submitted studies inc luded bacteriaproduced purified DvSnt7 dsRNA test material as well as plant tissue (grain) from MON 8741 I 

corn. Comparative analyses showed the equivalence o r the bacteri all y-prod uced and purified 

DvSnf7 dsRNA test material with the active ingredient expressed in MO 87411 com (U.S. 

EPA 20 15). Additionally, although it is recommended that non-target testing be conducted at a 

test dose at least I OX the EEC whenever possible, the test dose margin can be less than I OX 

where unce11ainty in the system is low or where high concentrations of test materi al are not 

possible to achieve due to test organism feeding habits. BPPD may also allow for testing at lower 

doses in cases where many species are tested or tests arc very sensitive, altho ugh the 

concentration used must exceed the EEC. 

Limit dose testing on representati ve organisms from several taxa was performed with DvSnf7 in 

support of the proposed MON 874 11 FIFRA Section 3 seed increase registration. A study to 

determine soil degradation of DvSnf7 has also been submiued. as well as a bioinformatics study 

and a Geld study of effects to nontarget arthropods in agricultural environments. Rationale to 

add ress other data requested, as well as a response to the 20 14 SAP report, were also submitted. 

The indi vidual results for nontargct organism and soil degradation testing for DvSnt7 dsRNA arc 

summarized in Table 1. The studies are described in more detail below, and full reviews of each 

study can be found in the individual Data Evaluation Records. 

To address data requirements for Cry3Bb I, Monsanto Company cited to studies with Cry3 Bb I 

expressed by other corn events (MO 88017, MON 863) that have been previously reviewed by 

5 
EPA. To allow for bridging to the Cry3Bbl data, information must be submitted to EPA to show 

that the protein expressed by MON 87411 is equi valent to the Cry3Bb I protein used in the cited 

studies, the protein is not expressed at levels higher than previously assessed, and that no 

synergism exists between the Cry3Bb 1 and DvSnf7 expressed in MON 874 1 I. EPA determi ned 

that the Cry3Bb I expressed in MON 87411 corn is biochemically and functionall y equivalent to 

the Cry3Bb1 expressed in MON 88017 corn, which is similarly equivalent to the Cry3Bbl 

expressed in MON 863 corn. The data submitted lo support the registration of Cry3Bbl in MON 

880 17 and MON 863 are discussed in the updated BRAD for those events (U.S. EPA 2010a). 

Concerning equivalence of protein expression levels. Cry3Bbl protein expression in MON 87411 

is comparable to or below that of MO 88017 and MON 863, as discussed above. Monsanto 

Company has additionally submitted two studies on synergism between Cry3Bbl and DvSnf7, 

whi ch is summari zed in the table and discussed further below. 

Table 1. Summary of data submitted for OvSnf7 dsRNA as expressed in MON 87411 corn 

to comply with data requirements published in 40 CFR & 158.2150. 

Data OPPTS Test Substance Results Summary and Classification MRID 

Requirement Guideline No. 

Avian dietary 885.4050 MON 87411 A 42-day dietary study showed no adverse 49315111 

testing. corn grain effects on survival, broiler performance, or 

broiler chicken, carcass yield in broiler chickens fed a diet 

Gallus containing MON 87411 grain. 

domes/ icus Classification: Suoolemental 

Av ian inhalation 885.4100 N/A Not required. Inhalation is not expected to be a NIA 

testing route by wh ich birds may be exposed lo DvSnt7 

dsRNA. 

Wild mammal 885.4150 IA Studies with laboratory animals are sufficient to 49505804 

testing determine risk to wild mammals. Adverse 49505806 

effects were not observed in a 28-day study with 

purified DvSnt7 dsRNA or in a 90-day study 

with MON 87411 com grain included in the diet 

at 33%. 

Freshwater fish 885.4200 MON 87411 In an 8-week study, consumption of MON 49505805 

testing. corn grain 874 11 cbrn grain at the exposure level tested 

channel catfish, (30% of the diet) had no adverse effects on 

lc:talurus survival, weight gain, or diet conversion. 

punc:talus Classification: Suonlemcntal 

Freshwater 885.4240 NIA Submitted rationale was adequate to conclude Contained 

invertebrate adverse effects are unlikely with the proposed within 

testing seed increase registration. 49553302 

Classifica tion: Acceota blc 

Estuarine and 885.4280 NIA Not required. Exposure in marine and/or NIA 

marine animal estuarine environments is not expected to be 

testing significant. 

Non-target plant 885.4300 NIA Not required. Exposure to nontarget plants is NIA 

testing expected to be minimal. 

Non-target insect 885.4340 Microbially- No adverse effects on survival, development to 493 15 11 4 

testing. lady produced adult stage, time to emergence, or adult biomass 

beetle, DvSnt7 dsRNA were observed in C. macu/a/G fed I 000 ng 

Coleomegilla Dv nf7 dsR A/g diet for 21 days. 

muc:ulata Classification: Supplemental but 

uol!radeable 

6 
Data OPPTS Test Substance Results Summary and Classification MRID 

Requirement Guideline No. 

Non-target insect 885.4340 Microbially- No adverse effects on adu lt surviva l in P. 493 15 115 

testing, parasitic produced foviolatus fed a 30% honey/water solution 

wasp. Pediohus DvSnf7 dsR A containing I 000 ng DvSnf7 dsR Alg diet in a 

.fiwiolatus 20-day study. 

Classification: Supplemen ta l but 

upgradeable 

1 on-target insect 885.4340 Microbially- o adverse effects on survival or rate of adult 49315117 

testing, insidious produced emergence in 0. i11sidios11s nymphs fed 1000 ng 

nower bug, DvSnf7 dsRNA DvSnf7 dsRNA/g diet in a I 0-clay study. 

Orius insidio.ws Classification: Supplemental but 

up!?.radcable 

on-target insect 885.4340 M icrobially- No adverse effects on survival or reproduction 49315118 

(soi I arthropod) produced were observed in F. candida exposed to I 000 ng 

testing, DvSnf7 dsRNA DvSnf7 dsRNA/g diet in a 28-day study. 

springtail. Classifica tion: Supplemental but upgradeable 

Folsomia 

c:andida 

Non-target insect 885.4340 M icrobially- No adverse effects on survival, deve lopment to 493 1511 9 

testing, carabid produced adult stage. time to emergence. or adult biomass 

beetle, Poeci/us DvSnf7 dsR A were observed in carabid beetle larvae fed I 000 

cha/cites ng DvSnf7 dsRNA/g diet in a 35-day study. 

Classifica tion: Supplementa l but 

uo!!radeable 

Honeybee 885.-1380 Microbially- o adverse effects were observed in survival or 49315112 

testing, larvae produced development of larvae provided a single dose 

Apis mell(fera DvSnf7 dsRNA equivalent to 11 .3 ng DvSnf7 dsRNA per bee. 

Classifica I ion: Acccota ble 

I loneybee 885.-1 380 Microbially- No adverse efTects on survival were observed 49315113 

testing, adults produced when honey bee adults fed on a sucrose solution 

Apis mell((era DvSnf7 dsRN/\ contai ning 1000 ng DvSnf7/g diet for 14 days. 

Classification: Supplemental but 

Un1>radea ble 

Earthworm 850.6200 Microbial ly- o adverse efTects on survival or percent change 493 15116 

toxicity, produced in biomass in earthworms exposed to 5000 ng 

Eisenia andrei DvSnf7 dsRNA DvSnl7 dsRN A/g artificial soil in a 14-day 

contact study. 

Classification: Supolcmental but uo2radcable 

Soil fate and 885.5200 Microbially- Based on Quantigene® 2.0 analysis of soil and 49315122 

degradation produced bioactivity measured in Southern corn 

DvSnf7 dsRNA rootworm, DvSnf7 dsRNA degrades rapid ly in 

Plus MO soil with 90% dissipa1ion time ranging 45 - 55 

87411 plant hr. 

tissue Classification: Acceptable 

A.I. interaction NIA Microbial ly- The toxicity ofCry3Bb 1 protein to L. 493 15121 

(synergism). produced decemlineato does not change when exposed 

Colorado potato DvSnf7 dsR A either to a lethal concentration ofCry3Bbl 

beetle, and Cry3 Bbl alone or simultaneously exposed to Cry3Bb I 

Leptinotarsa and I 000 ng DvSnf7 dsRNA/g diet. 

decemlineata Classification: Acccotable 

/\.I. interact ion NIA Microbially- No dilTerence in toxicity (LCso) was observed in 49505803 

(synergism), produced SCR exposed to a lethal concentration of 

Southern corn DvSnf7 dsRNA Cry3Bb I combined with a sublethal 

root worm. and Cry3Bbl concentration (EC ,0 for growth inhibition) of 

7 
Data OPPTS Test Substance Results Summary and Classification MRIO 

Req uirement Guideline No. 

Diahrmica DvSnf7 DsRNA or vice versa. Testing with 

11ndecimp1111ctata additional concentrations is necessary to 

howardi confirm the potential for synergism. 

Classification: Suoolementa l 

Field evaluation 850.2500 MON 87411 No evidence of adverse effects to non target 49553304 

of non target corn plants arthropod populations or communities were 

arthropod observed between MON 87411 corn plots and 

abundance genetically similar (except for transgenic traits) 

controls in field plots studied in the U.S.. Brazil, 

and Argentina. The data appear to be variable. 

and the study is not likely to predict differences 

among treatments. 

Classification: Supplemental 

Non target NIA DvSnf7 No exact ~ 21 nt matches were found when the 49553306 

organism Sequence 240 nt DvSnf7 sense strand sequence was 

bioinformatic queried against transcriptorne sequence 

analysis collections !Tom 23 nontarget organisms. 

Classification: Supplemental 

Non target NIA MON 87411 The document provides some information that is 49505802 

organism useful to the risk assessment, and information 

environmental was incorporated herein where pertinent. 

risk assessment Classification: Supplemental 

B. Study Summaries for DvSnn dsRNA 

1. Birds 

Avian Dietary Toxicity 

A 42-day feeding assessment using Cobb x Cobb 500 broilers (Gallus domesticus) was 

conducted to compare the nutritional value of diets containing MON 87411 maize grain and a 

near isogenic conventional maize control NL6169 with similar background genetics to MON 

87411. Five additional diets containing maize grain representatives of the population of 

commercial conventional maize varieties were included. Nutrient analyses of each maize grain 

source were used to formulate diets having identical nutrient specifications, which were fed to 

the broilers ad libitum throughout the study. Treatments were assigned to pens randomly within 

five blocks of 14 pens each (seven male and seven female) with ten broilers per pen for a total of 

70 pens and 700 birds. Broilers were weighed on Day 0 at the end of the study just prior to 

processing to assess carcass yield. Pen feed intake was determined for the duration of the study, 

and used to calculate feed to weight gain ratio. 

There were no biologically relevant differences in broiler performance. carcass yield, or 

mortality (mortality ranged from 0-0.8% across all treatments) between broilers fed diets 

containing MON 8741 I maize grain and those fed diets containing near isogeni c conventional 

control NL6 l 69 maize gra in. The study report does not state the percentage of grain in the diet; 

however. Monsanto Company's environmental risk assessment (MRID 49505802) states that the 

percentage of MON 87411 in the diet was 57%. While this is more than half of the diet, 

expression of DvSnf7 dsRNA in grain (0.104 x l 0-3 ug/g tissue dwt) is much lower than in other 

8 
plant tissues. This level of exposure is not a maximum hazard dose that is typicall y required fo r 

Tier I nontarget organism studies, but it is also unclear whether this amount is representative of 

the EEC, since birds may be exposed through routes other than consumption of corn grain (e.g., 

consumption of pest insects that have consumed leaf or orher tissue). Also, while the o ther 

vari ables measured (e.g., weights of various body parts) may indicate the nutritional performance 

of the grain, it is unclear what relevance they have to survival or reproduction of wild birds. 

While this study does provide some insight into the potential for adverse effects in birds, 

parti cularl y fo r extended ex posure periods (6 weeks), its usefulness in determining risk depends 

on the actual level of exposure to birds in the field. Because of this limitation, the study was 

determined to be supplemental. 

A vi an lnhalat ion Toxicity 

Avian inhalation toxicity is a conditional data requirement of 40 CFR 158.2150. Since EPA does 

not anticipate significant exposure of birds to DvSnt7 dsRNA via inhalation. these data are not 

required for registration of DvSnf7 dsRNA in MO 87411. 

2. Wild Mammals 

Two studies are available with which to assess the potential toxicity of DvSnf7 in wild 

mammals, including a 28-day acute oral toxicity study with mice (MRIDs 49505804 and 

49576 10 I) and a 90-day feeding study with rats (MRI Os 49505806 and 4957610 I). These 

studi es were reviewed by EPA and classified as acceptable (U.S EPA 20 15). In the 28-day study. 

mice were dosed daily with DvSnf7 test material with purity of I 00% a.i .. and no adverse effects 

were observed that were attributable to the test material. This study established a NOA EL of I 05 

mg/kg/day. In the 90-day study, rats were fed a diet of 33% grain, and no treatment-related 

effects were observed on mortality, clinical signs, functional observational battery. body weight, 

body we ight gain, food consumpti on, food efficiency, clinical pathology, o rgan weights, gross 

pathology. or microscopic pathology. Based on information described by Monsanto Company, 

the OAEL was determined to be 1899 and 2303 mg/kg total cage body weight per day for 

ma les and females, respectively: however, these values appear to describe the NOA EL for MON 

87411 in the di et, and not the NOAEL for DvSnf7 dsRNA. 

3. Freshwater Fish and Invertebrates 

Freshwater Fish Toxicity 

A dietary bioassay with channel catfish (lctalurus pwu:tatus) was conducted to address the Tier I 

data requirement for freshwater fish. This study was designed to evaluate the nutritional 

equi valence of diet conta ining maize grain from MON 874 11 compared to diets containing 

maize grain from a near isogeni c conventional control or from each of four reference maize 

vari eties. The diets were formulated to contai n 30% of the appropriate mai ze grain variety, to 

contain approximately 32 ± I% crude protein, and to be isonitrogeno us. Gro ups o f I 00 fish each 

(five replicates per group) were fed the appropriate diet daily for eight weeks. After e ight weeks, 

there were no statistically signifi cant differences among the treatment groups for food 

consumption, weight gain. or diet conversion. and grain from MO 874 1 I com was considered 

9 
to be nutritionally equivalent to conventional maize grains when fed to channel catfish. Whi le 

this information is helpful, this study has similar limitations for determining toxicity in 

freshwater fish as described for the broiler chicken study above. In this case, the diet consisted o f 

· just 30% of grain, in which re latively low levels of DvSnt7 are expressed compared to other com 

tissues. The study shows that adverse e ffects are not likely based on the study conditions, and 

this information is useful to the risk assessment. However, the certainty of risk estimated from 

this study is dependent on the actual exposure in the field. For this reason, the study was 

classified as supplemental. 

Due to uncertainties assoc iated with potenti al exposure in aq uatic systems to MO 8741 I plant 

material and unknown fa te of DvSnf7 in pl ant tissue, EPA requested information on degradation 

of DvSnJ7 in plant tissue in its 75-day defi ciency letter. EPA also stated that if DvSnf7 was 

shown to persist in plant tissue, then a Tier I acute freshwater fish study (OCSPP guideline 

885.4200) would be required to show no acute adverse effects. The catfish study above was 

submitted independently of this request, and had not been reviewed and considered at the time 

the letter was written. Monsanto Company responded to the request with rationale discussing the 

rapid degradation of R A and DvSnt7 dsR A. EPA· s previous conclusions regarding exposure 

to PlPs in aquatic environments, and lines of evidence leading to a conclusion of low likelihood 

of effects. including physiological baJTiers to exposure and results of bioinformatics analysis. 

This information is adequate to address potenti al effects to freshwater fish for the proposed seed 

increase registration. 

4. Marine and Estuarine Fish and Invertebrates 

EPA has previously determined that exposure to Br deri ved PlPs in marine and estuarine 

environments is not significant. At this time, there is no information to indicate that this 

assumptio n would not a lso apply to OvSnl7. Therefore, no data were required to address toxicity 

of marine or estuarine fish and invertebrates. 

S. Nontarget Plants 

While it is unclear at this point whether DvSnt7 may be present in root exudates of MON 87411 

com. it is likely that any DvSnt7 that enters the soil via this source or by release from plant 

tissues will rapidly break down (see discussion of soil degradation below). Therefore, significant 

exposure to plants is not expected, and data to address nontarget plant toxicity were not required. 

6. Nontargct Insects and Other Invertebrates 

Nontarget insects 

In a 35-day dietary toxicity study, carabid beetles (Poecilus cha/cites (Say]) were exposed to 

DvSnt7 test material at a nominal concentration of 1,000 ng/g of diet (MRI D 493 151 19). A 

water assay control and a potassium arsenate positive control were also used. Exposure of the 

carabid beetles to the three diets was replicated three times with 20 insects per diet for a total of 

60 insects per diet treatment. All dietary exposures were initiated with the first instar larvae. 

Survival. development to adult stage. developmental time (days) to adult emergence. and adult 

10 
biomass were measured. Mean percent survival at test termination for the test, control, and 

positive control diets were 93%, 92%, and 65% respectively. None of the larvae fed the positive 

control diet developed to the pupa stage. The lower survival and inhibited development of insects 

fed potassium arsenate confirmed that the test system was capable of detecting toxic effects 

through the dietary exposure; however, in most other studies test insects did not survive the 

positive control treatment. The percent development to adult for larvae exposed separatel y Lo test 

and assay control diets averaged 70% and 75%. For the test and assay control diet treatment 

groups. the development time to adult emergence averaged 33 days. Adults fed test and control 

diet treatments average biomass was 31.9 mg and 32.3 mg, respecti vely. The study report 

determined a NOEC of I ,000 ng/g of diet; however, the LCso is more typically determined for 

non target maximum hazard dose studies. The results of this study demonstrate that exposure to 

DvSnf7 _968 RNA at the maximum hazard dose concentration of I 000 ng/g diet had no adverse 

effect o n the survival, development and growth of the carabid beetle, Poecilus cha/cites. While 

the study does provide useful information about the potential toxicity of DvSnf7 in this species, 

the study was determined to be supplemental, but upgradeable if the following information is 

provided: 1) data showing typical mortality of P. cha/cites exposed to potassium arsenate or 

explanation of the rel ati vely high survi val in this group, 2) cumulative raw data on food 

consumption (or confirmation that larvae consumed all food between feedings), mo rtality, 

development time, and raw data on adult biomass. These data are needed to confirm the results 

of the study. These data must be provided for use of this study to support a full commercial 

registration of MO 8741 I. 

Toxicity of DvSnf7 was also determined in insidious flower bug (Orius insidiosus) nymphs in a 

study involving I 0 days of exposure (MRID 49315117). The insects were allowed to feed ad 

libitum on a diet containing OvSnt7 at I 000 ng/g diet. A positive control diet containing l 00 μg 

of potassium arsenate per gram diet and a negative control diet were also included. Each 

treatment consisted of 40 individually housed nymphs, which were initially exposed as 5-day old 

nymphs. Survival and development were observed. Survival of the test nymphs was 93% in both 

assay control and OvSnf7 treatment. Development to adult occurred at 95% and 98% in the assay 

control and Ov nf7 treatment. respectively. The average development time for test nymphs to 

deve lop into adults was I I. I I ± 0.15 days in the assay control treatment and I 0.87 ± 0.13 days in 

the DvSnf7 968 R A treatment. In contrast the 0. insidiosus nymphs fed the potassium arsenate 

diet showed_ I 00% morta lity by day 1 O with I 3% of nymphs developing to adults. The results of 

this study show that continuous dietary exposure to DvSnf7 for 10 days at a concentration of 

I 000 ng/g diet had no effect on the survival and development of 0. insidious nymphs. The 

results of this study are useful to the risk assessment, but the study is classified as supplemental. 

but upgradeable because data are needed to confirm the results. Data required include: a) the 

length of time that observations were made beyond the 10-day exposure, b) raw data on 

mo11ality, emergence. and development time, and c) food consumption data or clarification as to 

whether food was consumed completely between feedings. These data must be provided for use 

of this study to support a full commerci al registration of MON 8741 I. 

Larvae of the lady beetle, ColeomegiL/a maculata (Coleoptera: Cocinellidae), were a lso tested in 

a 21-day study to determine the potential toxicity of DvSnf7 to nontarget insects (MRI D 

493 I 51 14). Larvae were exposed to DvSnf7 in a test diet at a nominal concentration of 1000 

ng/g of diet. A negative water control and a positive control of potassium arsenate were also 

11 
tested. Exposure to C. maculata to the three diets was replicated three times with 20 individually 

housed insects per diet replicate for a total of 60 insects per diet treatment. All dietary exposures 

were initiated with the first instar larvae, and the study was continued until all surviving control 

and test insects developed lo the adult stage. Survival, development to adult stage, development 

time (days) to adult emergence, and adult biomass were observed. Mean percent survival at test 

tennination for the test, negative control. and positive control diets were 92%, 90%. and 17%. 

respectively. None of the larvae fed the positive control diet developed to the pupa stage. The 

percent development to adult for larvae exposed separately to test and negative control diets 

averaged 92% and 90%, respectively, and development time to emergence for both treatment 

groups was 15 days. Adult biomass for test or control diet averaged l 0.2 mg for both treatments. 

The OEC was determined to be l 000 ng /g of diet. since that was the concentration tested. The 

results of this study demonstrate that exposure to DvSnt7 at the maximum hazard dose 

concentration of I 000 ng/g of diet had no adverse effect on the survival, development, and 

growth of this species. These results are useful to the risk assessment, but the study was 

classified as supplemental, but upgradeable, because confirmatory information was not available. 

Data needed to upgrade the study include: I) raw data on cumulative mortality, development 

time, percent development to adult, and adult biomass for each replicate for each treatment, and 

2) food consumption data and/or confirmation that food was consumed completely between 

feedings. These data must be provided for use of this study to support a full commercial 

registration of MON 87411. 

A fourth species, parasitic wasp (Pediobiusfoveolatus) was also tested for toxi city to DvSnt7 in 

a 20-day study. P.foveolatus were exposed to OvSnt7 test material at 1000 ng/g in a 30% 

honey/water (v/v) solution (MRID 49315115). An assay control treatment of 30% honey/water 

solution and a positive control diet at 200 μgig potassium arsenate in a 30% honey/water solution 

were also included. The treatments were replicated four times with I 0 adult wasps per replicate 

for a total of 40 wasps per treatment. All dietary exposures were initiated with newly emerged 

adults after approximately 24 hours of acclimation, and feeding ad libitum was allowed for the 

duration of the study. There was no mortality of P. fove olatus adults that had fed the OvSnt7 

treatment diet or the assay control diet after 20 days. In contrast, the P. foveolatus adults fed the 

potassium arsenate treatment diet showed 100% mortality at day 12. The results of this study 

demonstrate that continuous dietary exposure to DvSnt7 for 20 days at a concentration of 1000 

ng/g diet had no effect on the survival of the parasitic wasp, P. foveolatus. The NOEC of the 

DvSnf7 for the parasitic wasp was l 000 ng/g diet; however, since the study was designed more 

like a limit test, the LCso would be the typical endpoint expressed from this type of study. The 

study is useful to the risk assessment, but was determined to be supplemental, but upgradeable, 

since confirmatory information was missing. Additional information required includes: l) 

explanation for a discrepancy in a sub-report describing samples tested for test material stability. 

2) clarify the use of specific gravity in calculating the nominal dose, and 3) food consumption 

data or clarification that the wasps consumed all food between feedings. These data must be 

provided for use of this study to support a full commercial registration of MON 87411. 

Honey Bee 

Two studies with honey bees were submitted to support the registration of MON 87411, 

inc luding a stud y with adults (MRID 49315113) and another with larvae (MRID 49315112). The 

12 
study with adults evaluated the potential dietary effects or DvSnt7 on survival in a 14-day 

continuous feeding study. Newly emerged adults(~ 2 day old) were exposed to DvSnt7 at a 

concentration of I 000 ng/g, an assay control (no treatment), and a positive control (potassium 

arsenate at 20 μg/ml), each of which was present in a 50% sucrose/purified water (w/v) solution. 

Each treatment group included 80 adult bees in four replicates of 20 bees per repl icate (one 

replicate per cage). Each cage was provided with approximately 10 ml of the appropriate 

treatment diet solution and bees were allowed to feed ad libitum. Bees from each treatment group 

were observed daily for mortality, abnormal behavior, and appearance. No adverse effects on 

survival, behavior and appearance were observed, and a NOEC for this study was determined to 

be I 000 ng of DvSnf7 test material/g diet according to the study report. This study was classified 

as supplemental but upgradeable. because food consumption data and/or clarification as to 

whether the diet was completely consumed by the bees between feed ings was not provided. 

These deficiencies do not affect the usefulness of the study for this proposed registration, but 

these data must be provided for use of this study to support a full commercial registration of 

MO 87411. 

ln a 17-day dietary toxicity study, honey bee larvae were exposed to a single dose of DvSnt7 test 

material prepared at a concentration of 1000 ng/g and delivered in a I 0 μI aliquot of 30% (w/v) 

sucrose/purified water to each larval cell for a total mass of 11.3 ng DvSnf7/cell. Additional 

treatment groups included an assay control (30% sucrose solution) and a positive control (30% 

solution containing potassium arsenate at 2,000 μg/ml). Larva (2 to 3 days old) in all groups 

were exposed to a single 10 ~ti dose of the appropriate treatment at study initiatio n and observed 

over the course of larval/pupal development and adult emergence. Each treatment group 

consisted of 80 honey bee larvae in four replicates of20 honey bee larvae per replicate. There 

was I 00% survival in honey bee larvae treated with either DvSnf7 or the assay control. 

Emergence of adult honey bees from the test larvae in both the DvSnl7 and the assay control was 

initiated on the same day (Day 14), approximately 50% on Day 15, and reached I 00% on Day 17 

in both treatments. There was no survi val in the positive control confirming the validity of the 

exposure system. Observations of newl y emerged adult bees indicated no adverse behavioral or 

morphological effects. Thus, the NOEL for this study was determined to be 11.3 ng of DvSnf7 

per larva, based on the study report. The LDso is more typicall y reported in nontarget studies 

designed to test a maximum hazard dose. This study is consistent with similar studies accepted 

by EPA in the past. and is classified as acceptable. 

Soil Invertebrates 

In a 14-day contact study. ten adult earthworms(£. andrei) were exposed to a single appli cation 

of DvSnf7 incorporated into an artificial soil substrate at a rate equivalent to 5000 ng R A per g 

soil dry weight. No food was provided during the duration of the test. The test treatment was 

compared to a control treatment of purified water added to the soil. Mortality was assessed over 

the 14-day testing period, and the change in fresh weight of the worms was assessed for 

survivors at 14 days after treatment. There was no observed mortality a fter 14 days. The mean 

percentage change in biomass was -9.4% and -8.4% in the control groups and treatment group, 

respectively. This study was classified as supplemental but upgradeable. This guide line (OECD 

207) is intended to test toxicity to earthworms via contact exposure. It is unclear whether this 

route of exposure is appropriate for toxicity testing with double stranded R A. and a dietary 

13 
study may have been more appropriate for a test with this organism. Also, it is unclear whether 

the DvSnt7 test materi al would have degraded during the 14 day exposure period, and there is no 

assurance that the concentration in the soil was consistent throughout the study. The guideline 

a lso states specifically that multiple concentrations must be tested to establish a NOEC. The 

study could be upgraded with justification that the exposure route is relevant, and that the 

DvSnf7 was stable throughout the duration of the study. These data must be provided for use o r 

this study to support a ful l commercial registration of MON 87411. 

Toxicity to soil invertebrates was also tested with a 28-day dietary toxicity study of DvSnt7 to 

springtails (Folsomia candida). DvSnt7 test material was incorporated into an inactivated yeast 

medium diet with a concentration of l 000 g DvSnf7 per gram of diet. This test diet was 

compared to an untreated control diet of inacti vated yeast, a blank (no food) diet, and a toxic 

reference diet (inactivated yeast treated with the insect growth regulator, teflubenzuron). 

Springtails were allowed to feed ad libitum. At 28 days, there were no significant effects on the 

survival or reproductive capacity of the DvSnt7 treatment group compared to controls. Mortality 

in the untreated control. blank diet, and reference diet was 0%, 0%, and 93%, respectively. The 

mean number of progeny in the treatment groups was 169 for the untreated control diet, 0 for the 

blank diet, 167 for the DvSnt7 diet, and 0.3 for the reference diet. The NOEC, based on 

mortality, was 1000 ng DvSnt7 per gram of diet according to the study report. However, since 

the study is designed more like a limit or maximum hazard dose study, more typical endpoints 

determined from these data are the LCso and ECso. This study is useful to this risk assessment, 

but was determined to be supplemental, but upgradeable, since confirmatory data were not 

provided. Data that are required to upgrade the study include: 1) data on food consumption or 

confim1ation that all food was consumed between feedings, and 2) raw data on mortality and 

reproduction. These data must be provided for use of this study to support a full commercial 

registration of MON 87411. 

Field Evaluation ofArthropod Abundance 

A field study (MR!D 49553304) was conducted to determine whether MON 87411 maize 

produces adverse effects on nontarget insect populations across three geographic locations: 

United States, Argentina, and Brazil. Four to six sites were chosen within each geographic 

location, and within each site MON 87411 and a conventional control (with similar genetic 

background except for the insecticidal and herbicide tolerant traits) were tested for differences in 

nontarget arthropod abundance and nontarget pest damage. Each site included plots with MO 

874 l l. the conventional control, and four conventional reference maize varieties (which varied 

across sites). At each site, nontarget arthropod abundance was determined using both sticky traps 

and visual counts, and counts were conducted multiple times over the growing season. Pest 

damage was also assessed using methods specific to each pest, and damage was assessed several 

times over the growing season. Out of 121 individual site comparisons, only five significant 

differences were detected between MON 87411 and the conventional control in nontarget 

arthropod abundance. For pest damage, 56 comparisons were made, and significant differences 

between MON 874 11 and the conventional control were detected in three. These differences 

were determined not to be biologically significant. 

14 
This sludy provides some informalion on polential effects lo arthropods in a more realistic lield 

setting; however, the study design presents uncertainties in the data, which limits conclusions 

that can be drawn. It is unclear that the study, as designed. would detect differences and provide 

strong indication that adverse effects do not occur in the field. Limitations include: 

I) Abundance data are represented as counts of arthropods at the taxonomic level of order or 

fami ly, and some of these taxa include many species that likely vary in their ecological 

functions and intra-community relationships. Natural variation in species abundance may 

also exist, and differences in sensitivity to the treatment may also vary among species. 

Pooling them removes the ability to account for such variation in the data, and increases 

the difficulty of detecting treatment effects. 

2) Plot sizes were relatively small, and it is unclear whether the plots were immediately 

adjacent or separated within the site. This is important for understanding the 

independence of the data between treatments, since more mobile arthropods could move 

between plots with different treatments. It is also unclear whether the plots were in the 

vicinity of other corn that might serve as sources of the insects observed in the study. 

This is important to know, because if there were adverse effects that would otherwise 

reduce counts within MON 87411 plots, they may be replaced by other migrating insects 

that could end up being counted in the census. 

3) Mean counts that were determined to be significantly different between MON 87411 and 

the controls are compared to the range of means determined within the reference plots. In 

most cases it appears that the means for MO 8741 I and the control plots are consistent 

with the reference ranges. However, in some cases both means for MON 87411 and the 

conventional control are outside the reference range and in others the range is very wide. 

This may bring into question the reliance on the reference mean ranges as a check against 

the test for statistical significance, and may be an indicator of general vari ation in the data 

that makes it di fticult to detect differences among treatments. 

4) Pest damage could indicate that certain nontarget species are not affected by the 

insecticidal substances in MON 87411, especially since the insects are known to have fed 

on the plants. However, the values presented are low in some cases, and it is unclear 

whether enough data were collected to detect differences among treatments. The study 

report indicates that pesticides were appli ed in the study. and it is unclear how this may 

have affected the results. 

Clarification of some of the above points would be helpful in the evaluation of this study. The 

data do provide some indication that adverse effects to nontarget arthropods may not be highly 

likely. However, the results are relatively weak compared Lo a hypothesis driven fi eld study 

conducted at an appropriate scale, and a field study with a more robust design would be needed 

to demonstrate unequivocally a lack of effects in the field. For this reason, this study was 

detem1ined to be supplemental. 

7. Degradation In Soil 

A laboratory study was performed to determine the aerobic degradation rate or the DvSnt7 RNA 

in soil (M RID 49315122). Microbially-produced DvSnt7 test material or a control substance was 

incorporated into soi ls collected from three different states in the U.S .. Illinois (IL), Missouri 

15 
(MO) and North Dakota (ND). The DvSnf7 treatment also included MON 87411 leaf material. 

The USDA soil classifications were Silt Loam (IL), Loamy Sand (MO) and Clay Loam (ND). 

The soils differed in their physicochemical characteristics (silt, sand and clay composition, pH, 

organic matter, etc.) based on a pre-study analysis. Soil samples were extracted and analyzed for 

DvSnf7 concentrations using QuantiGene® 2.0 kits obtained from Affimetrix, Inc. [n addition, 

the decline in the insecticidal activity was quantified by measuring the mortality of insects 

exposed to extracts of the soil sampl es, using a di et-incorporation insect bioassay with the 

southern com rootworrn. The estimated DTso and DT90 values for degradation of DvSnf7 _968 in 

soi l based on QuantiGene molecular analysis were the following: DTso of 19 hr, 28 hr, and 15 hr 

and DT9o of23 hr, 35 hr, and 22 hr for IL, MO, and NO, respectively. The decline in biological 

activity of DvSnf7 in soil, measured against standard concentration curves in the SCR bioassay, 

produced estimated DTso and OT9o values of 18 hr, 29 hr, 14 hr and 23 hr, 34 hr, and 2 1 hr for 

IL, MO, and ND respectively. The bioassay data were also examined using a standard 3-

parameter logistic equation to characterize the decline in insect mortality against incubation time, 

without conversion to soil concentrations. This approach produced DvSnf7 dissipation curves 

comparable to those obta ined from the standard concentration analyses with QuantiGene and 

insect bioassay. ln this evaluation, the estimated time to 50% dissipation of insect mortality was 

23 hr, 33 hr, and 22 hr, and the 90% dissipation time was 55 hr, 45 hr, and 45 hr for IL, MO, and 

ND, respectively. 

Results from the soil degradation study indicate that DvSnf7 _968 degraded within approximately 

2 days after application to soil, regardless of texture, pH, clay content and other soil differences, 

as measured by molecular analysis (QuantiGene) and by insecticidal activity (bioassay). 

Therefore, the study supports the conclusion that the dsRNA transcript deri ved from DvSnt7 

suppression cassette of MON 87411 is unlikely to persist or accumulate in soil under conditions 

similar to those simulated in the experiment at levels above analytic detection. There was a low 

level of biological activity beyond the time that the dsRNA degraded below the quantification 

limits of the QuantiGene analysis. It is unknown whether the residual activity was a result of the 

DvSnf7 that was added to the soil, or the release of DvSnf7 from degrading plant tissue. Hence, 

there is some uncertainty regarding the persistence of OvSnf7 RNA from MON 87411 at low, 

but biologically active levels. Further investigation would be needed to confirm that a low level 

of biological activity does not continue beyond two days; however, given the rapid degradation, 

any residual activity is not expected to continue for very long. This study was determined to be 

acceptable. 

8. Synergism 

As discussed above, the potential for synergistic effects must be evaluated to allow bridging to 

data previously submitted on Cry3Bb 1. Two studies were submitted to address this requirement. 

Synergism in SCR 

One study was submitted that tested the potential for synergism in the sensitive insect species, 

southern corn rootworm (MRID 49505803). The study was conducted as a 12-day bioassay. The 

potential for interaction between DvSnf7 and Cry3 Bbl was evaluated by comparing the LC so 

values of a lethal level of Cry3 Bb 1 in the presence of DvSnf7 at a fixed sub-lethal concentration 

16 
and a lelhal level of DvSnl7 in lhe presence o r Cry3 8b l at a tixed sub-lethal concentration. The 

fixed sub-l ethal concentration for DvSnf7 and the Cry3Bb 1 protein used in the two binary 

mixtures was equi valent to an EC10 value for growth inhibition dete1mined in method 

developmenl bioassays. Cry3 Bb I and Ov nl7 were also lested at lethal levels a lone (not as part 

of a binary mixture). Assay and buffer contro ls were included, and mortality was low (7% -

11 %). The SCR concentrati on responses for the Cry3 Bbl prote in alone and the Cry3 Bbl prote in 

in the presence of a fixed sub-lethal concentration of OvSnf7 were nearl y identical and this is 

reflected by comparable LC so values for the Cry3Bb I protein alone and the binary mixture 

(LCso= 46.28 μ g/ml di et for Cry3Bbl alone and 40.65 μg/ml for Cry3Bbl combined with 

DvSnt7 at a fixed sub-lethal concentration). The SCR concentration responses for the DvSnf7 

alone and DvSnf7 combined with Cry3Bb 1 at a fixed sub lethal concentrati on were also very 

comparable (LCso= 6.35 ng/ml diet for OvSnf7 alone and 8. 16 ng/ml diet for DvSnf7 combined 

with the sub letha l concentration of Cry3 Bb I). The study authors concluded that these data show 

a lack or synergism between DvSnf7 and CryJ Bb l. 

EPA had previo usly raised concerns about the use of the fixed sub lethal concentration that was 

stated to be the limit at which overt toxic signs would be observed. Monsanto Company justified 

this approach by citing to studies in which fixed sublethal concentrations have been used 

successfull y to assess for and characterize synergistic interacti on, including Tabashnik ( 1992) 

and citati ons within, as well as Belden and Lydy (2006). These papers do describe experiments 

in which fixed subletha l concentrations have been used, even in very small amounts (e.g .. 

Macintosh ct al. [ 1990), c ited in Tabashnik [ 1992]). However. these papers also describe 

additiona l testing at multi ple concentrations. determination or synergism based on o bservati ons 

of sublethal effects, and/or adjustments in the target mortality in the component present in higher 

concentrati on (e.g., reduced to 10-20% in Mac intosh et al. (1 990]). These approaches better 

characteri ze the nature of the potential interacti on, and ensure that an effect would be observed i r 

it were to occur. While the approach used in this study is not unlike some of these o ther studi es, 

and may be valid, there is still uncertainty as to whether a synergistic effect on mo rta lity coul d be 

observed at the sub lethal levels of DvSnf7 or Cry3Bb I tested. Additional testing using subl ethal 

endpoints (e.g., growth inhibition) or testing both Cry3Bbl and DvSnf7 at multiple 

concentrations or median lethal levels would reduce this uncertainty. T his study is classifi ed as 

supplementa l, and is sullicicnt to support the proposed seed increase registration. However. an 

additiona l study better designed to detect synergism between Cry3 Bb 1 and DvSnf7 is required to 

support a full commercial registration for ION 874 11. 

Synergism in Colorado Potato Beetle 

An additional confirmatory study was submitted to evaluate the potentia l for interactio n between 

the OvSnf7 and Cry3Bb l protein using Colorado potato beetle (CBP, Leplinotarsa 

decemlineata) in a 12-day continuous feeding bioassay (MRIO 49315 12 1 ). The Colorado potato 

beetl e is not susceptible to DvSnf7, according to Bachman, et al. (20 13). The po tentia l for 

interacti on was evaluated by comparing the LC so values and the 95% confidence intervals of the 

Cry3 Bb I protein in the presence and absence of DvSnf7 at a fi xed concentrati on of 1000 ng/m L 

diet. Nominal test concentrati ons for the Cry3 Bbl protein were based on the results of previous 

bioassays performed at the testing facility. Mean mortality in all assay and buffer contro ls was 

I 0.42% at assay terminati on. Colorado potato beetl e demonstrated nearl y identical 

17 
concentration-effect relationships to the Cry3Bb l protein alone and in the presence of fixed 

concentration of DvSnf7. The study is acceptable as a confirmatory study of no interaction in a 

nontarget species, but does not definitively test interaction because it is unclear whether CPB has 

any sensitivity to OvSnf7. 

9. Nontarget Organism Bioinformatic Analysis 

At the request of EPA, Monsanto Company submitted a bioinformatic evaluation of the DvSnt7 

sequence, wherein the 240 nt DvSnf7 sequence was compared to DNA transcriptome databases 

for 23 nontarget organisms (MRID 49553306). The evaluation was conducted using STELLAR 

software version 1.3 (Kehr et al. 2011) to identify exact 21 nucleotide matches between the 

DvSnt7 query and sequences contained in collections for the nontarget organisms, which were: 

Anas platyrhynchos (mallard), Apis mellifera (honey bee), Bombus terrestris (bumble bee), Bos 

taurus (cattle), Caenorhabditis elegans (nematode), Canisfamiliaris (domestic dog), Columba 

livia (rock pigeon), Danaus plexippus (monarch butterfly), Dania rerio (zebra fish), Daphnia 

pulex (water flea), Drosophila melanogaster (common fruit fly), Equus caballus (horse), Gallus 

gallus (red junglefowl/chicken), Hyalella azteca (scud), Locust migratoria (migratory locust), 

Megachile rotundata (alfalfa leafcutter bee), Mus musculus (house mouse), Nasonia vitripennis 

(jewel wasp), Oryzias latipes (medaka), Rattus norvegicus (Norway rat), Solenipsis invicta (red 

fire ant), Sus scro.fa (pig), and Tetranychus urticae (spider mite). No exact matches of 21 nt or 

greater were found with any of the sequences searched. 

This bioinformatic analysis provides some additional information toward another line of 

evidence of no expected adverse effects in non target organisms. Of these, the honey bee and 

chicken have also been tested against exposure to DvSnf7, and no adverse effects were identified 

in those studies. The report for the bioinformatic evaluation does not provide details about how 

these species vvere chosen, nor does it include searches of other coleopterans that would be more 

closely related to the target species. Bioinformatics analyses are not considered to be predictive 

of effects. EPA is currently evaluating how bioinformatics may be used in nontarget risk 

assessments, and at this time this analysis is considered to be supplemental information. 

V. Risk Characterization and Description 

A. Risk of Nontarget Organisms to Cry3Bbl Expressed in MON 87411 Corn 

Monsanto Company has cited to studies previously submitted to support the registration of MON 

88017 corn. MON 88017 expresses a Cry3Bbl protein that is equivalent to the Cry3Bbl 

expressed in MON 87411. As discussed above, EPA previously determined that the Cry3Bbl 

expressed in MON 88017 is equivalent to the Cry3Bb l expressed in MON 863, and much of the 

data submitted to support MON 863 was bridged to support the registration of MON 88017. 

Expression of the Cry3 Bb I protein in MON 87411 does not exceed that of either MON 863 or 

MON 88017. The synergism study conducted with a sensitive test species does not show 

definitively that a synergistic relationship does not exist between the Cry3Bb1 and DvSnf7 in 

MON 87411, and additional data on synergism is required to support a full commercial 

registration for MON 87411. However, the data available plus preliminary data provided from an 

additional study on growth inhibition in com rootworm (described in MRID 49505802) indicate 

18 
that synergism is unlikely. Therefore, bridging to data to support registrations or these other 

events expressing Cry3Bb I is appropriate for this registration. 

Acceptable data cited showed that adverse effects are not expected to occur for birds, wild 

mammals, freshwater fish and invertebrates, honey bee (Apis mell{fera), nontarget insects 

(including Monarch butterflies) and soil invertebrates. Additional supplemental studies on the 

insect activity spectrum, a preliminary Tier IV field evaluation, and summaries of various higher 

tier laboratory and field studies were also submitted. These data were sufficient lo allow EPA to 

determine that the Cry3Bb I expressed in MO 88017 corn presented minimal risk to nontarget 

organisms. Therefore, adverse effects are similarly not expected to result from exposure to the 

Cry3Bbl expressed in MON 8741 I. 

EPA also concluded that adverse effects to federally listed threatened and endangered ("listed") 

species were not anticipated, and made a "no effect" conclusion for direct and indirect effects to 

I isted species and their designated critical habitats (U.S. EPA 20 I 0). These assessments have 

focused on listed coleopteran species, due to the specificity ofCry3Bbl to insects of this order. 

Since the 20 10 assessment, an additional coleopteran species, Casey's June beetle (Dinacoma 

caseyi), has been listed. EPA does not anticipate any effects to this species because it is I imited 

to specific habitats, primarily desert wash areas with sandy soils, which would not be provided in 

areas developed for growing corn. Development (primarily urban or suburban) leading to 

destruction of habitat is cited as one of the reasons for decline of this species and its current 

restricted range (U.S. FWS 20 11 ), and areas where soil is highly disturbed do not provide good 

habitat (U . . FWS 2013). Additionally. any adverse effects to nontarget organisms are expected 

to be limited to the field in which MON 8741 l is grown, since pol len is not expected to move 

significantly fa r from the corn field (U.S. EPA 2010b) and expression in poll en is very low 

compared to other plant tissues. Significant pollen drift is unlikely in areas where the species 

may occur; therefore. exposure is not anticipated. Therefore. EPA makes a "no effect'. 

determination for direct and indirect effects for Casey's June beetle and its designated critical 

habitat for Cry3Bb I expressed in MON 87411. 

B. Risk of Non target Organisms to OvSnn Expressed in MON 87411 Corn 

1. Birds and Mammals 

EPA typically does not quantify exposure of nontarget vertebrates to PIPs, and instead relies on 

toxicity testing that is conducted at exposure levels reasonably expected to equal or, preferably, 

exceed maximum exposure levels in the field based on expression studies. Corn grain is the plant 

tissue that birds and mammals are most likely to consume, so ir exposure assumptions are limited 

to grain only. then exposure is relatively simple to understand. However. it is likely that all 

species of birds and mammals that inhabit corn agroecosystems are exposed via other sources. 

While incidental dietary exposures to other corn tissues may occur, exposure through 

consumption of pest insects or other invertebrates insensitive to the toxins produced by MON 

8741 1 corn is another route by which birds and mammals may be exposed lo OvSnf7. While 

OvSnf7 is not expected necessarily to accumulate within invertebrates, other tissues potentially 

consumed by these invertebrates show expression of DvSnf7 at levels several orders of 

magnitude higher than in grain. While the level or exposure may not be significant, the extent of 

19 
exposure via this source, or any other possible source, has not been determined. A "worst case" 

scenario that would reduce uncertainty would be based on the highest concentration shown to be 

expressed in the PIP plant based on expression studies submitted to EPA. Ideally, exposure in 

Tier I nontarget organism testing would be derived from these levels. 

Current toxicity data available to assess risk for birds consists of a 6-week study of broiler 

chickens fed 57% MON 87411 grain. If this level of exposure is representative of maximum 

exposures in the field, then this study is adequate to address potential effects in birds over the 

period of time observed. As discussed above, determination of actual exposure may be 

complicated, and consumption of grain does not represent a "worst case" exposure with complete 

certainty. Even if consumed, physiological barriers exist in birds that significantly limit uptake of 

DvSnt7, including salivary and other digestive ribonucleases (Park et al. 2006, Stevens and 

Hume 1995) and acidic gut environments (Akhtar 2009, Loretz et al. 2006, O'Neill et al. 2011 ). 

Bioinformatic analyses with Gallus gal/us and Columba Livia indicated no exact 21 nt matches 

with the DvSnt7 sequence, providing an additional line of evidence toward expectation of no 

effects (EPA is cautious, however, in interpreting this information as predictive of effects). 

Based on these lines of evidence, adverse effects in birds are unlikely. For a larger scale 

commercial release of MON 874 11 , however, a confirmatory feeding study with birds should be 

submitted. 

Data avai lable for mammals includes a 28-day study with mice, in which mice were dosed daily 

at levels up to I 05 mg/kg/day, and a 90-day study with rats, which were fed MON 87411 corn 

grain at 33% of the diet for the duration of the study. Both studies indicated no adverse effects. 

The dose of 105 mg/kg/day likely exceeds maximum short term exposure levels to mammals that 

would occur in the field, and 90-day exposure to MON 87411 grain provide some insight into 

longer term exposures at low levels. As with birds, other lines of evidence are available to 

supplement the data provided, including physiological barriers as discussed above, and 

bioinformatics analysis with mammalian species. Based on these lines of evidence, the lack of 

effects observed in the available toxicity studies, and the limits on the proposed seed increase 

registration, adverse effects to wild mammals are not anticipated to result from the proposed 

registration of OvSnf7 dsRNA in MON 87411. 

It is noted that EPA has requested a study examining the stability of OvSnf7 dsRNA in whole 

blood to confinn rationale that blood components would break down any dsRNA that may enter 

the mammalian bloodstream. Should these data show that DvSnf7 dsRNA persists in the blood 

such that effects could be considered likely to occur, EPA will require additional effects testing 

with vertebrate species. 

2. Freshwater Fish and Invertebrates 

Exposure in aquatic environments may occur as a result of pollen drift to these areas, and also 

potentially through leaf or other post-harvest crop residue movement off of cultivated fields. 

EPA previously concluded that exposure to Cry protein PlPs in aquatic systems is limited. In the 

response to the deficiency letter requesting data on DvSnf7 breakdown in plant tissue, Monsanto 

provided a discussion of the studies supporting this conclusion and the low likelihood of 

20 
exposure to senescent com plant tissue in aquatic environments. This information is sufficient to 

suppo rt this assumption for the proposed seed increase registration of MO 1 8741 I. 

As discussed above, the channel catfish study provided some information on the potential dietary 

toxicity of DvSnf7 to freshwater fish, but that the usefulness of this study is limited by the 

assumptions of exposure. This study showed that adverse effects as observed arc not expected 

under similar exposure conditions. Since exposure in aquatic environments is assumed to be 

minimal based on EPA's current assumptions for Cry protein PIPs, then this study likely is 

suffic ient to capture potential acute or subchronic adverse effects of DvSnt7 to freshwater fish at 

realistic environmental exposure levels. Exposure is also expected to be reduced by the proposed 

acreage and time limitations on the registration. and expected rapid breakdown of dsR A in 

soils. Additionally, EPA notes that expression of DvSnf7 generally declines over the growing 

season, and is several orders of magnitude lower in corn stover and senescent root. Therefore, 

levels of DvSnf7 available in corn plants post harvest are expected to be lower compared to the 

growing season. Based on all of this information, adverse effects to freshwater fish are not 

expected to occur as a result of the proposed seed increase registration for DvSnf7 in MON 

87411. Similarly, since exposure is not expected to be significant in aquatic environments for 

this proposed registration, adverse effects on freshwater invertebrates are not anticipated. 

3. Marine and Estuarine Fish and Invertebrates 

Significant exposure to OvSnf7 is not expected in marine and estuarine environments. Therefore, 

adverse effects are not anticipated for fish or invertebrates in these environments. 

4. Nontarget plants 

Exposure of nonlarget plants to DvSnf7 is not expected to be signifi cant as discussed above. 

Therefore, adverse effects to nontarget plants are not anticipated. 

5. Nontarget Insects and Other Invertebrates 

Monsanto Company has presented an extensive discussion of the specificity of Dv nf7 for its 

target pest. Western com rootworm. and its close relative. Southern com rootworm. Primary 

work to demonstrate specificity for the target pest is described in Bachman et al. (20 13). In this 

study, bioassays were conducted with either DvSnf7 dsRNA or dsRNA developed from an 

ortholog of the S11f7 gene in several close re latives. This study showed that DvSnf7 was only 

active within the Chrysomelidae Family of coleopterans. which provides a strong line of 

evidence toward the specificity of DvSnf7. 

Exposure to nontarget insects for PIPs is typicall y based on expression levels within plant 

tissues. Expression levels can differ between plant tissues and also over time. and the plant tissue 

with the most relevance to the insect diet is typically used to determine dosing levels or diet 

concentration in nontarget testing. Expression levels in pollen are often used, since many 

non target insects will consume pollen, and expression in leaf may also be considered. As 

described above for birds and mammals, there is some uncertainty with the actual exposure in the 

fi eld given the varying diets within arthropod communities. Therefore. a conservative approach 

21 
to estimating exposure of nontarget organisms to PIPs would be to utilize the highest expression 

levels measured among all tissues based on dry weight measurements. While this may 

overestimate exposure for certain organisms, it reduces uncertainty around actual exposure in the 

field . 

In the environmental risk assessment submitted by Monsanto Company, NOEC values 

determined from nontarget testing were compared to maximum expected environmental 

concentrations (MEECs) to calculate margins of exposure (MOEs). If MO Es are > 1 Ox the 

MEEC, then risk is assumed to be negligible. Utilizing the highest mean dry weight expression 

level measured (84.8 x I 0·

3 μg/g whole plant tissue from V3-V4 development stage), NOEC 

values of I 000 ng/g dete1mined in nontarget insect testing would produce a MOE of 11.8, which, 

if using this approach, would indicate a low likelihood of adverse effects. The MOE for bees 

would be >3,000, assuming the maximum recorded expression in pollen represents the MEEC 

for honey bees (maximum dw value measured was 0.292 x 10·3 μg/g). This approach has been 

found to be acceptable by EPA in previous risk assessments; however, given that the studies are 

limit dose tests in which only one concentration was tested, there is uncertainty around the 

biological meaning of the NOEC. Without testing at additional higher concentrations, it is not 

possible to know how close the NOEC is to levels at which effects that might be observed. 

Nonetheless, EPA considers level at which toxicity was tested to be sufficiently above expected 

environmental concentration, and that the studies submitted are reliable tests of potential effects 

to nontarget insects at realistic field exposure levels. 

Monsanto Company supplemented the above data with a discussion of bruTiers to exposure in 

invertebrate species. It is clear from some studies of dsRNA in invertebrates that some 

physiological barriers exist that can significantly limit uptake in insects. These barriers include 

ribonucleases in saliva (Allen and Walker 2012, Christiaens et al. 2014) and DNA/RNA nonspecific nucleases in the hemolymph (Garbutt et al. 2013). Other evidence of some barriers are 

demonstrated in studies requiring certain conditions for delivery of dsRNA, such as by 

microinjection (Whyard et al. 2009) or nanoparticle encapsulation (Sarathi et al. 2008). While 

these baniers are not fully understood, and information is not yet available to extrapolate 

between insect taxa, it is apparent that such barriers reduce bioavailability in some insects. 

Based on the specificity of the intended effect, inve1tebrate studies submitted, and other 

information provided from the literature, EPA concludes that adverse effects to nontarget insects 

and other terrestrial invertebrates are not expected to occur as a result of the registration of 

OvSnt7 in MON 87411 as proposed. Honey bees and other pollinators are included in this 

conclusion, and the results of testing with both larvae and adult honey bees provide strong 

evidence in support. Additionally, Monsanto Company has provided results of bioinformatics 

analyses with both honey bee and bumble bee, neither of which identified exact 21 nt matches 

with DvSnt7. As noted above, these data provide supplemental information to support this 

con cl us ion, but EPA recognizes that they are not necessarily predictive of effects. 

The potential for off-target and other unintended effects, which were discussed as an uncertainty 

by the SAP, appears to be unlikely based on the data and other information provided. 

Understanding completely all of the potential physiological mechanisms providing barriers to 

uptake or leading to unintended effects cannot be known without extensive research, which is 

22 
beyond the scope of EP I\ ·s ecological risk assessments. However, EPA previously requested 

testing to address potential effects on reproduction in two additional insect species. Off-target 

and other unintended effects orDvSnf7, if they occur, are expected to be more evident in insects, 

since they are more close ly related to the target pcsl. Therefore, thorough testing to ensure that 

all portions of the li fe cycle are not affected would provide an additional line of evidence to 

confirm that unintended effects of dsRNA would not occur in the insect species tested had these 

observations been made. These data should be submitted to support a Full commercial 

registration of MO 8741 I. 

6. Outcrossing and Weediness 

EPA has previously determined that there is no signifi cant risk of gene capture and expression of 

any Bt endotoxin by wild or weedy relati ves of corn in the U.S., its possessions or territories (see 

extensive discussion in U.S. EPA 20 I 0). Since these conclusions are based on the nature or 

pollination, survival of hybrid offspring, and weediness in com and its relatives, these 

conc lusions a lso apply to DvSn t7. 

7. Risk Assessment Conclusions for DvSnf7 

Based on the data and rationale presented, adverse effects to nontarget organisms are not 

expected as a result of the proposed seed increase registration of DvSnl7 expressed in MON 

874 11 com. Additional confirmatory data as described above will be required to support a full 

commercial registration for M ON 8741 I. 

EPA agrees that the intended effect of downregulation of the Sn.fl gene is I ike ly o nly to be 

observed in the target insect and close relati ves. Therefore, conc lusio ns for listed spec ies as 

determined for Cry3 Bb I would also apply to DvSnf7, since the target insect is a coleopteran 

species. Monsanto Company has provided an extensive d iscussion to show that the rea listic 

potential for off-target e ffects to other species is likely to be very low, and EPA has requested 

confirmatory data to support this conclusion. 

C. Risk of Nontarget Organisms to C1113Bb l and DvSnf7 dsRNA Combined as 

Expressed in MON 87411 Corn 

ince expressio n levels of Cry3Bbl do not exceed those previously considered in ecological risk 

assessments, and because prcl iminary data indi cate a lack of synergism bet ween these Pf Ps, the 

conclusions for Cry3Bb 1 and DvSnf7 individuall y also apply to the combination of these P!Ps in 

MON 8741 I for this seed increase registration. 

VI. References 

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Targeting 17: 49 1-495. 

23 
Allen, M.L. and W.B. Walker III. 2012. Saliva of Lygus lineolaris digests double stranded 

ribonucleic acids. Journal of Insect Physiology 58: 391 -396. 

Bachman, P. M., R. Bolognesi, W.J. Moar, G.M. Mueller, M.S. Paradise, P. Ramaseshadri, J. 

Tan, J.P. Uffman, J. Warren, B.E. Wiggins, and S.L. Levine. 2013. Characterization of the 

spectrum of insecticidal activity of a double-stranded RNA with targeted activity against 

Western corn rootworm (Diabrotica virg!fera virg{fera LeConte). Transgenic Research 22: 

1207-1222. 

Belden, J.B. and M.J. Lydy. 2006. Joint toxicity of chlorpyri fos and esfenvalerate to fathead 

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Christiaens, 0 ., L. Sv.1evers and G. Smagghe. 2014. dsRNA degradation in the pea aphid 

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Macintosh, S.C., G.M. Kishor, F.J. Periak, P.G. Marrone, T.B. Stone, S.R. Sims, and R.L. Fuchs. 

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O'Neill, M.J., L. Bourre, S. Melgar an C.M. O' Driscoll. 201 1. Intestinal delivery of non-viral 

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203-2 18. 

Park, M.J., Y. Li, T. Yu, B.M. Brinkman and D.T. Wong. 2006. Characterization of RNA in 

saliva. Clinical Chemistry 52: 988-994. 

Sarathi, M., M.C. Simon, C. Venkatesan and A.S. Hameed. 2008. Oral administration of 

bacterially expressed VP28dsRNA to protect Penaeus monodon from white spot syndrome 

virus. Marine Biotechnology 10: 242-249. 

Stevens, C.E. and I.D. Hume. 1995. Digestion of carbohydrate, lipids, and protein and the 

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Digestive System. Cambridge University Press, Cambridge, United Kingdom. 

24 
Tabashnik .. B.E. 1992. Evaluation of synergism among Bacillus thuringiensis toxins. Applied and 

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U.S. Environmental Protection Agency (U.S. EPA). 20 10a. Biopesticides Registration Action 

Document: Bacillus thuringiensis Cry3 Bb I Protein and the Genetic Material Necessary for 

Its Production in MON 863 Corn; Bacillus thuringiensis Cry3Bb I Protein and the Genetic 

Material Necessary for Its Production in MON 88017 Corn. Biopesticides and Pollution 

Prevention Division, Office of Pesticide Programs. 

hup:I'""'" .epa.!!m lpestici<les/biopcsricidcs/rips/crdbb 1-brnd.p<lr. 

U.S. EPA. 20 I Ob. Biopesticides Registration Action Document: Cry I Ab and Cry IF Bacillus 

thuringiensis (Bt) Corn Pl ant-Incorporated Protectants. Biopesticides and Pollution 

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hllp://v\WW.cpa.!!()\'/()ppbppd I /biopcsticiclcs/pips/crv I r-crv I ab-brad.pd I". 

U.S . EPA. 2015. Memorandum from J. Facey to J. Kausch, Subject: Review or Product 

Characterization and toxicity data in support for a seed increase Sec. 3 Registration of PlantIncorporated Protectant (PIP) MO 1 87411 corn, dated August I 0. 20 15. 

U.S. Fish and Wildlife Service (U.S. FWS). 2011. Endangered and Threatened Wildlife and 

Plants; Determination of Endangered Status of Casey's June Beetle, and Designation of 

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hllp:l/ccos.f\vs.!!O\ /docs/rcco\'cn· plan/C.113 Rcco,·cn Outline Fl 11\l..p<lf". 

Whyard .. S. A.D. Singh and S. Wong. 2009. Ingested double-stranded R As can act as specicsspeci fic insecticides. Insect Biochemistry and Molecular Biology. 39: 824-832. 

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